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p53 Protocols pp 165-181 | Cite as

p53 Actions on MicroRNA Expression and Maturation Pathway

  • Hiroshi I. Suzuki
  • Kohei MiyazonoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 962)

Abstract

The tumor suppressor p53 orchestrates multiple cellular pathways as a central node of anti-oncogenic programs in response to DNA damage, oncogene activation, and several stresses. In addition to the principal role as a transcription factor that transactivates many target genes involved in apoptosis and cell cycle control, p53 has been shown to exert various transactivation-independent effects both in the nucleus and in the cytoplasm. Diversity of p53 activities is further emphasized by the recent studies revealing the close interaction between the p53 and microRNA (miRNA) world. We recently demonstrated that p53 promotes the processing of several primary miRNA transcripts through association with Drosha, a central RNase III in miRNA biogenesis, under DNA damage-inducing conditions. In contrast to wild-type p53, cancer-derived p53 mutants attenuate miRNA maturation. These findings reveal a novel aspect of p53 activities and suggest complex crosstalks between miRNA biogenesis and intracellular signaling pathways. In this chapter, we describe the methods for evaluation of the effects of p53 on miRNA expression, an interaction between pri-miRNA and Drosha complex, and pri-miRNA processing activity of the Drosha complex.

Key words

microRNA p53 p68 Drosha Dicer Processing 

Notes

Acknowledgments

We thank K. Yamagata and S. Kato for technical advice on in vitro processing analysis. This work was supported by KAKENHI (Grant-in-Aid for Scientific Research) and the Global Center of Excellence Program for “Integrative Life Science Based on the Study of Biosignaling Mechanisms” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. H.I.S. is supported by a research fellowship of the Japan Society for the Promotion of Science for Young Scientists.

References

  1. 1.
    Brown CJ, Lain S, Verma CS, Fersht AR, Lane DP (2009) Awakening guardian angels: drugging the p53 pathway. Nat Rev Cancer 9:862–873PubMedCrossRefGoogle Scholar
  2. 2.
    Soussi T, Beroud C (2001) Assessing TP53 status in human tumours to evaluate clinical outcome. Nat Rev Cancer 1:233–240PubMedCrossRefGoogle Scholar
  3. 3.
    Green DR, Kroemer G (2009) Cytoplasmic functions of the tumour suppressor p53. Nature 458:1127–1130PubMedCrossRefGoogle Scholar
  4. 4.
    Suzuki HI, Miyazono K (2010) Dynamics of microRNA biogenesis: crosstalk between p53 network and microRNA processing pathway. J Mol Med 88:1085–1094PubMedCrossRefGoogle Scholar
  5. 5.
    Krol J, Loedige I, Filipowicz W (2010) The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 11:597–610PubMedGoogle Scholar
  6. 6.
    Mendell JT (2005) MicroRNAs: critical regulators of development, cellular physiology and malignancy. Cell Cycle 4:1179–1184PubMedCrossRefGoogle Scholar
  7. 7.
    Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N et al (2004) The Microprocessor complex mediates the genesis of microRNAs. Nature 432:235–240PubMedCrossRefGoogle Scholar
  8. 8.
    Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J et al (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415–419PubMedCrossRefGoogle Scholar
  9. 9.
    Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK et al (2006) Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125:887–901PubMedCrossRefGoogle Scholar
  10. 10.
    Fukuda T, Yamagata K, Fujiyama S, Matsumoto T, Koshida I, Yoshimura K et al (2007) DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs. Nat Cell Biol 9:604–611PubMedCrossRefGoogle Scholar
  11. 11.
    Yi R, Qin Y, Macara IG, Cullen BR (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17:3011–3016PubMedCrossRefGoogle Scholar
  12. 12.
    Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R (2005) Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123:631–640PubMedCrossRefGoogle Scholar
  13. 13.
    Suzuki HI, Miyazono K (2011) Emerging complexity of microRNA generation cascades. J Biochem 149(1):15–25PubMedCrossRefGoogle Scholar
  14. 14.
    He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y et al (2007) A microRNA component of the p53 tumour suppressor network. Nature 447:1130–1134PubMedCrossRefGoogle Scholar
  15. 15.
    Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, Lee KH et al (2007) Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 26:745–752PubMedCrossRefGoogle Scholar
  16. 16.
    Raver-Shapira N, Marciano E, Meiri E, Spector Y, Rosenfeld N, Moskovits N et al (2007) Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol Cell 26:731–743PubMedCrossRefGoogle Scholar
  17. 17.
    Tarasov V, Jung P, Verdoodt B, Lodygin D, Epanchintsev A, Menssen A et al (2007) Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle 6:1586–1593PubMedCrossRefGoogle Scholar
  18. 18.
    Braun CJ, Zhang X, Savelyeva I, Wolff S, Moll UM, Schepeler T et al (2008) p53-Responsive micrornas 192 and 215 are capable of inducing cell cycle arrest. Cancer Res 68:10094–10104PubMedCrossRefGoogle Scholar
  19. 19.
    Georges SA, Biery MC, Kim SY, Schelter JM, Guo J, Chang AN et al (2008) Coordinated regulation of cell cycle transcripts by p53-Inducible microRNAs, miR-192 and miR-215. Cancer Res 68:10105–10112PubMedCrossRefGoogle Scholar
  20. 20.
    Yan HL, Xue G, Mei Q, Wang YZ, Ding FX, Liu MF et al (2009) Repression of the miR-17-92 cluster by p53 has an important function in hypoxia-induced apoptosis. EMBO J 28:2719–2732PubMedCrossRefGoogle Scholar
  21. 21.
    Suzuki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S, Miyazono K (2009) Modulation of microRNA processing by p53. Nature 460:529–533PubMedCrossRefGoogle Scholar
  22. 22.
    Pothof J, Verkaik NS, van Ijcken IW, Wiemer EA, Ta VT, van der Horst GT et al (2009) MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response. EMBO J 28:2090–2099PubMedCrossRefGoogle Scholar
  23. 23.
    Bates GJ, Nicol SM, Wilson BJ, Jacobs AM, Bourdon JC, Wardrop J et al (2005) The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor. EMBO J 24:543–553PubMedCrossRefGoogle Scholar
  24. 24.
    Park SY, Lee JH, Ha M, Nam JW, Kim VN (2009) miR-29 miRNAs activate p53 by targeting p85 alpha and CDC42. Nat Struct Mol Biol 16:23–29PubMedCrossRefGoogle Scholar
  25. 25.
    Le MT, Teh C, Shyh-Chang N, Xie H, Zhou B, Korzh V et al (2009) MicroRNA-125b is a novel negative regulator of p53. Genes Dev 23:862–876PubMedCrossRefGoogle Scholar
  26. 26.
    Fornari F, Gramantieri L, Giovannini C, Veronese A, Ferracin M, Sabbioni S et al (2009) MiR-122/cyclin G1 interaction modulates p53 activity and affects doxorubicin sensitivity of human hepatocarcinoma cells. Cancer Res 69:5761–5767PubMedCrossRefGoogle Scholar
  27. 27.
    Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R et al (2006) A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124:1169–1181PubMedCrossRefGoogle Scholar
  28. 28.
    Mudhasani R, Zhu Z, Hutvagner G, Eischen CM, Lyle S, Hall LL et al (2008) Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells. J Cell Biol 181:1055–1063PubMedCrossRefGoogle Scholar
  29. 29.
    Su X, Chakravarti D, Cho MS, Liu L, Gi YJ, Lin YL et al (2010) TAp63 suppresses metastasis through coordinate regulation of Dicer and miRNAs. Nature 467:986–990PubMedCrossRefGoogle Scholar
  30. 30.
    Chen C, Tan R, Wong L, Fekete R, Halsey J (2011) Quantitation of microRNAs by real-time RT-qPCR. Methods Mol Biol 687:113–134PubMedCrossRefGoogle Scholar
  31. 31.
    Niranjanakumari S, Lasda E, Brazas R, Garcia-Blanco MA (2002) Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods 26:182–190PubMedCrossRefGoogle Scholar
  32. 32.
    Sun BK, Deaton AM, Lee JT (2006) A transient heterochromatic state in Xist preempts X inactivation choice without RNA stabilization. Mol Cell 21:617–628PubMedCrossRefGoogle Scholar
  33. 33.
    Ni JQ, Liu LP, Hess D, Rietdorf J, Sun FL (2006) Drosophila ribosomal proteins are associated with linker histone H1 and suppress gene transcription. Genes Dev 20:1959–1973PubMedCrossRefGoogle Scholar
  34. 34.
    Gregory RI, Chendrimada TP, Shiekhattar R (2006) MicroRNA biogenesis: isolation and characterization of the microprocessor complex. Methods Mol Biol 342:33–47PubMedGoogle Scholar
  35. 35.
    Ishizuka A, Saito K, Siomi MC, Siomi H (2006) In vitro precursor microRNA processing assays using Drosophila Schneider-2 cell lysates. Methods Mol Biol 342:277–286PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of Molecular Pathology, Graduate School of MedicineUniversity of TokyoTokyoJapan

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